LEUKOCYTE MicroRNAS FOR USE IN DIAGNOSIS AND TREATMENT OF ENDOMETRIOSIS

ABSTRACT

Methods for diagnosis and treatment of endometriosis are described. Methods utilize the recognition that leukocyte miRNAs can be dramatically dysregulated subjects suffering from endometriosis. Accordingly, leukocyte miRNAs, as well as polynucleotides encoding the miRNAs can be utilized in the diagnosis and treatment of endometriosis.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 61/955,519 titled “Leukocyte MicroRNAs asBiomarkers for Endometriosis” of Nagarkatti, et al. filed on Mar. 19,2014, the disclosure of which is incorporated by reference herein.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under P91AT003961awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Endometriosis is an estrogen-dependent, inflammatory disease affectingapproximately 176 million women worldwide. It occurs when cells from thelining of the uterus grow in other areas of the body. It usually leadsto pain, irregular bleeding, and infertility.

It has been reported that immune dysregulation may play a role inendometriosis (Rier and Yeaman 1997). For instance, animal modelingstudies have indicated that lymph node immune cell populations includingT cells, dendritic cells and B cells were increased and endometrialstromal cells were significantly increased in lymph nodes from animalswith induced endometriosis (Hey-Cunningham, Fazleabas et al. 2011).

In addition, it has been documented that cytokines may play a criticalrole in immune cell differentiation, growth and functions (St Georgievand Albright 1993; Bryant, Ma et al. 2007; Steppich, Moog et al. 2007).Alterations in cytokine expression associated with inflammation havelong been observed in individuals with immune-mediated diseases (Feng,Scheinberg et al, 2012; Hu and Hwang 2012). Cytokines may bedysregulated in patients with endometriosis (Hou, Sun et al. 2009),suggesting that cytokines may play a role in endometriosis.

Epigenetic events are crucial in the control of both normal cellularprocesses and abnormal events associated with cancer (Feinberg, Ohlssonet al. 2006) and other human diseases (Egger, Liang et al, 2004). Majorepigenetic mechanisms include DNA methylation, covalentpost-translational modifications of histone proteins, and RNA mediatedgene silencing such as via small interfering RNA (siRNA) and microRNA(miRNA). It has been reported that miRNAs, small noncoding RNAs thatregulate gene expression by binding to complementary target mRNAs andinhibiting their translation, have been shown to play a crucial role inthe regulation of cytokine genes (O'Connell, Kahn et al. ; Xiao andRajewsky 2009), and are involved in the production and functions ofimmune cells. The specific role of some individual miRNAs has beendemonstrated in hematopoiesis and immune cell regulation. For example,miR-223 is involved in human granulopoiesis, erythropoiesis and B celldifferentiation (Malumbres and Lossos 2010), miR-155 promotes Th2 celldifferentiation (Rodriguez, Vigorito et al. 2007), and miR-326 promotesTh17 cell development (Du, Liu et al. 2009).

miRNAs have also been implicated in immune regulation. For example,miR-146a regulates the immune response to bacterial infections andmiR-181a modulates the sensitivity of T lymphocytes to antigens byregulating expression levels of various phosphatases from the T-cellreceptor signaling (Anglicheau, Muthukumar et al. 2010).

Recent studies indicate that microRNAs may play a role Ira endometriosis(Hawkins, Creighton et al. 2011; Wang, Zhao et al. 2013). Elucidation ofroles of specific microRNA dysregulated in endometrial patients couldhave great implications in understanding the pathogenesis ofendometriosis and developing diagnostic markers and therapeutic targetsfor endometriosis.

What are needed in the art are treatment methods for endometriosis thatinclude early detection of the disease state.

SUMMARY

According to one embodiment, a method for diagnosing and treatingendometriosis in a subject is disclosed. For instance, a method caninclude analyzing a test sample to determine a quantity of a leukocytemicroRNA in the test sample (i.e., either a mature microRNA or apolynucleotide that encodes the mature leukocyte microRNA). The methodcan also include comparing the quantity of the microRNA in the testsample to a control quantity of the leukocyte microRNA. Morespecifically, a determination that the quantity of the leukocytemicroRNA in the test sample differs from the control quantity by afactor of about 2 or greater can be indicative of endometriosis in thesubject. The method can further include administering a treatment forendometriosis to the subject.

According to another embodiment, a method for diagnosing and treatingendometriosis is disclosed. For instance, a method can include analyzinga test sample for the overexpression in the subject of one or morecytokines selected from IL-17, IFN-α, IL-4, and IL-10 by determining inthe sample the quantity of a leukocyte microRNA, the leukocyte microRNAsbeing microRNA-150a, microRNA-146a, microRNA-505, microRNA-320a, andlet-7a microRNA precursor or combinations thereof. The method can alsoinclude comparing the quantity of the microRNA in the test sample to acontrol quantity of the leukocyte microRNA. More specifically, adetermination that the quantity of the leukocyte microRNA in the testsample differs from the control quantity by a factor of 2 or greater canbe indicative of endometriosis in the subject. The method can furtherinclude administering a treatment for endometriosis to the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the change in immune cell populations in peripheralblood mononuclear cell (PBMC) samples from endometrial patients ascompared with controls.

FIG. 2 presents data concerning M1 (FIG. 2A) and M2 (FIG. 2B)macrophages in peritoneal huffy coat samples from endometriosispatients.

FIG. 3 graphically illustrates increased plasma level of IL-4 inendometrial patients as compared with control subjects and cytokinelevels in peritoneal fluid, cervical swab and uterine lavage samplesfrom endometrial patients.

FIG. 4 graphically illustrates increased plasma level of IL-10 inendometrial patients as compared with control subjects and cytokinelevels in peritoneal fluid, cervical swab and uterine lavage samplesfrom endometrial patients.

FIG. 5 graphically illustrates increased plasma level of IL-17 inendometrial patients as compared with control subjects and cytokinelevels in peritoneal fluid, cervical swab and uterine lavage samplesfrom endometrial patients.

FIG. 6 graphically illustrates increased plasma level of IFN-γ inendometrial patients as compared with control subjects and cytokinelevels in peritoneal fluid, cervical swab and uterine lavage samplesfrom endometrial patients.

FIG. 7 graphically illustrates increased plasma level of TNF-α inendometrial patients as compared with control subjects and cytokinelevels in peritoneal fluid, cervical swab and uterine lavage samplesfrom endometrial patients.

FIG. 8 presents miRNAs exhibiting up-regulation in PBMC from endometrialpatients as compared to controls.

FIG. 9 presents miRNAs exhibiting down-regulation in PBMC fromendometrial patients as compared with normal controls.

DETAILED DESCRIPTION

The following description and other modifications and variations to thepresent invention may be practiced by those of ordinary skill in theart, without departing from the spirit and scope of the presentinvention. In addition, it should be understood that aspects of thevarious embodiments may be interchanged both in whole or in part.Furthermore, those of ordinary skill in the art will appreciate that thefollowing description is by way of example only, and is not intended tolimit the invention.

The present disclosure is generally directed to the diagnosis andtreatment of endometriosis. More specifically, the present disclosure isdirected to recognition that the dysregulation of leukocyte microRNAs isindicative of endometriosis, and that this recognition can be utilizedin the diagnosis and/or treatment of endometriosis. For example, in oneembodiment, determination of the upregulation (overexpression) ordownregulation (underexpression) of leukocyte microRNAs can be utilizedin a protocol for treatment of endometriosis. In another embodiment,determination of the upregulation or downregulation of certain leukocytemicroRNAs can be utilized to determine the overexpression of certaininflammatory cytokines, which can be utilized in diagnosis and/ortreatment of endometriosis.

According to the present disclosure, it has been determined thatleukocyte miRNAs can be dramatically dysregulated in test samples ofsubjects suffering from endometriosis. For example, leukocyte miRNAs canbe dramatically dysregulated in the peripheral blood mononuclear cellsin subjects suffering from endometriosis. Accordingly, determination ofthe level in a test sample of mature leukocyte miRNAs and/orpolynucleotides encoding the mature miRNAs, can be utilized in thediagnosis and treatment of endometriosis. Techniques for miRNAexpression analysis in test samples are known in the art and reliableand as such the disclosed methods have a great potential in clinicaldiagnosis and treatment for endometrial patients.

As used herein, the term “test sample” generally refers to a biologicalmaterial suspected of containing a microRNA of interest or apolynucleotide encoding a microRNA of interest. The test sample may bederived from any biological source, such as a physiological fluid,including, blood, interstitial fluid, saliva, ocular lens fluid,cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasalfluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses,amniotic fluid, semen, and so forth. In addition, a solid materialsuspected of containing a microRNA or a polynucleotide encoding themicroRNA may be used as the test sample. The test sample may be useddirectly as obtained from the biological source or following apretreatment to modify the character of the sample. For example, suchpretreatment may include preparing plasma from blood, diluting viscousfluids, and so forth. Methods of pretreatment may also involvefiltration, precipitation, dilution, distillation, mixing,concentration, inactivation of interfering components, the addition ofreagents, lysing, etc. Moreover, it may also be beneficial to modify asolid test sample to form a liquid medium or to release the microRNA.

In addition, it should be understood that any polynucleotide encoding amicroRNA is encompassed herein including genes encoding microRNA (e.g.,MIR-xxx, in which xxx is the numerical reference to the particularmicroRNA), primary microRNA transcripts (pri-mRNA, e.g., mir-xxx),precursor microRNA transcripts (pre mRNA, e.g., mir-xxx), as well asmature microRNA (mRNA; e.g., miR-xxx). As used herein, the term “gene”generally refers to a locatable region of a genomic sequence that isinvolved in producing a microRNA, and includes regulatory regions,introns, transcribed regions and/or other functional sequence regions.

In determination that leukocyte miRNAs may be used as diagnostic markersand therapeutic targets for endometriosis, a number of peripheral blood,peritoneal fluid and endometrial tissues samples from patients diagnosedwith endometriosis have been examined. The data demonstrate thatleukocyte miRNAs may be used as diagnostic markers, for instance intreatment protocols, and also as therapeutic targets for endometriosis.

Flow cytometry has been used to analyze changes in immune cells in theperipheral blood mononuclear cell (PBMC) populations from patients withendometriosis. Analysis demonstrated that CD4+ T cells in the PBMC weresignificantly decreased in endometriosis patients compared to controls(FIG. 1). However, natural killer (NK) cells were dramatically increasedin endometriosis cases compared with normal donors (FIG. 1). In huffycoat samples from peritoneal fluid of endometriosis patients, there weresignificantly higher levels of CD14+ macrophages, especially M2macrophages (FIG. 2). These results suggested that immune dysregulationin the patients with endometriosis may play an important role inendometriosis development.

Cytokines mediate and control immune and inflammatory responses. Commonhuman diseases such as allergies, autoimmune diseases, chronicinfections, cancer and sepsis are characterized by a dysregulation inthe pro- versus anti-inflammatory cytokine balance. Cytokine analysisindicates that anti-inflammatory cytokines such as IL-4 (FIG. 3) andIL-10 (FIG. 4) as well as pro-inflammatory cytokines including IL-17(FIG. 5), IFN-γ (FIG. 6) and TNF-α, (FIG. 7) are significantly increasedin the plasma of endometriosis subjects, suggesting that specificcytokines play a role in endometriosis pathogenesis.

Based on in silico studies, it has been determined that both miR-150 andmiR-146a can lead to suppressed IL-17 production, miR-505 can inhibitIFN-γ gene expression, miR-320a can inhibit IL-4 and let-7e can inhibitIL-10. In addition, in accord with the present disclosure, it has beendetermined that these miRNAs (miR-150, miR-146a, mild-320a, miR-505 andlet-7e) can be dramatically down-regulated in women with endometriosis.Thus, in endometriosis patients, the decreased expression of thesemiRNAs may be responsible for the increased expression of these pro- andanti-inflammatory cytokines and this immune dysregulation may be usefulin detection and treatment of the pathophysiology in endometriosis.

According to one embodiment, detection of the down-regulation of one ormore of the microRNAs microRNA-146a, microRNA-505, microRNA-150,microRNA-320a, and let-7e can be indicative of the over expression ofthe related cytokines IL-17, IFN-γ, IL4 and IL-10 and thus diagnosis andtreatment of endometriosis.

Determination of the down-regulation of the microRNAs can be carried outaccording to any suitable detection scheme. In general, determinationcan include assessment of the concentration of the microRNAs in a testsample and comparison of that concentration with a normal control value.As used herein, the term “normal” with regard to concentration of acompound in a test sample generally refers to the average concentrationof the compound in a number individuals not affected by endometriosis.For example, the average concentration of the compound in about 10 ormore Individuals not affected by endometriosis.

Hybridization-based methods for determining the concentration of thetargeted miRNA can be used. Hybridization-based methods are direct assaymethods that do not necessarily require the amplification of RNAsamples. Hybridization-based methods can include, without limitation,Northern blot, microarray, and bead-based flow cytometry. The basicprinciple of these technologies is to hybridize probes with micro-RNAcontained in the sample and then perform the signal detection.

As used herein, the term “probe” generally refers to any molecule thatis capable of binding to a specifically intended target molecule, e.g.,a mature microRNA, Probes can be either synthesized or derived fromnatural materials. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic monomers.

As is known in the art, “hybridization” methods involve the annealing ofa complementary sequence to a target nucleic acid sequence (the sequenceto be analyzed). The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology. Hybridizationencompasses, but is not be limited to, slot, dot and blot hybridizationtechniques.

Quantitative real-time PCR (qRT-PCR) can also be utilized to determinethe concentration of miRNA in the sample. By way of example, one of twoqRT-PCR methods for miRNA expression analysis can be used: the poly(A)method (Shi, R. (2005) Biotechnique, 39, 519-525) or the stem-loopmethod (Chen, C. (2005) Nucleic Acids Res, 33, 1-9). The poly(A) methoduses a poly(A) tail added to the 3′ end of each mature miRNA by poly(A)polymerase. The tailed miRNAs are then subjected to reversetranscription (RT) using a universal RT primer containing 2 to 3degenerate nucleotides at 3′ end followed by an oligo(dT) and universalreverse primer sequence. The synthesized cDNA is amplified with specificforward and universal reverse primers. This method provides a simple andcost-effective method for miRNA assay.

The stem-loop method uses a designed stem-loop RT primer that containssix specific bases at its 3′ end that are complementary to the 3′ end ofthe specific mature miRNA for reverse transcription. The primer forreverse transcription has a stem-loop at its 5′ end, which enhances theaffinity of miRNA/DNA and avoids annealing of primer and pri- orpre-miRNA.

As utilized herein, the term “primer” refers to an oligonucleotide whichis capable of acting as a point of initiation of synthesis when placedunder conditions in which primer extension is initiated. Anoligonucleotide “primer” can occur naturally, as in a purifiedrestriction digest or be produced synthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

Optionally, a detection scheme can include concentrating or isolatingthe miRNA from a sample. For instance, a method can utilize commerciallyavailable anion exchange materials to isolate the microRNA of a sample.Either strong or weak anion exchangers may be employed. By utilizingselected solutions for adsorption and elution, the miRNA of a sample canbe purified, concentrated, and substantially isolated.

By employing a solution at known ionic strength for the initial bindingof the miRNA to the anion exchange column materials, most of the watersoluble components including other electronegative molecules such asproteins (weakly-bound contaminants) can be washed through the column.For elution, the required ionic strength is reached by using knownconcentrations of a salt such as NaCl, which may be mixed with a bufferto control pH, ideally corresponding to the lowest ionic strength atwhich the microRNAs will completely elute. Contaminating substancesbound to the anion exchange resin with higher stringency than thenucleic acids can thus be retained within the column.

A weak exchanger is one in which primary, secondary, or tertiary aminegroups (i.e., protonatable amines) provide exchange sites. The strongbase anion exchanger has quaternary ammonium groups (i.e., notprotonatable and always positively charged) as exchange sites. Bothexchangers can be selected in relation to their respective absorptionand elution ionic strengths and/or pH for the MiRNA being separated. Thesolution strengths are higher than the binding strengths.

According to one embodiment, analysis of a sample can be carried out asfollows:

Step 1: Obtain a sample (e.g., a peripheral blood sample) from asubject.

Step 2: Isolate immune cells (e.g., peripheral blood mononuclear cells)from the sample.

Step 3: Purify total RNA samples (including miRNAs) from the immunecells.

Step 4: Perform miScript reverse transcription to convert total RNA intoeDNA.

Step 5: Carry out quantitative real-time PCR using specific miScriptprimer assay to determine expression level of specific MiRNA in thesample.

Step 6: Compare the expression level of the targeted MiRNA in thesubject with normal control valued.

Step 7: Depending upon the comparison, diagnose the subject with and/ortreat the subject for endometriosis.

Disclosed methods are not limited to the down-regulated microRNAmentioned above. For instance, studies have also demonstrated thatmicroRNA-1225-5p, microRNA-572, microRNA-219-1-3p, microRNA-675 andmicroRNA-886-5p can be significantly up-regulated in subjects diagnosedwith endometriosis. Accordingly, in one embodiment, disclosed methodsfor treatment of endometriosis can include determining the expressionlevel of one or more of microRNA-1225-5p, microRNA-572,microRNA-219-1-3p, microRNA-675 and microRNA-886-5p, optionally inconjunction with one or more of the down-regulated microRNA discussedabove, e.g., microRNA-150, microRNA-146a, microRNA-320a, microRNA-505and let-7e.

Disclosed methods are not limited to the microRNAs mentioned above.Rather, endometriosis treatment methods disclosed herein are directed toleukocyte microRNAs that are up-regulated or down-regulated such thatthe quantity of the leukocyte MiRNA (or the polynucleotide that encodesthe leukocyte miRNA) in a test sample differs from that of a controlquantity by a factor of 2 or greater, or higher in other embodiments,for instance by a factor of 3 or greater, a factor of 4 or greater, or afactor of 5 or greater in some embodiments.

Examples of leukocyte microRNAs that have been determined to besignificantly (i.e., by a factor of 2 or greater) up-regulated ordown-regulated in subjects suffering from endometriosis as compared tothe expression levels for control subjects are provided in FIG. 8 andFIG. 9.

In one embodiment, a method can include determination that theconcentration of one or more of the up-regulated microRNAs of FIG. 8(microRNA-1246, microRNA-1225-5p, microRNA-223, microRNA-451,microRNA-572, microRNA-143, microRNA-219-1-3p, microRNA-92b,microRNA-148a, microRNA-450a, microRNA-593, microRNA-510,microRNA-1224-5p, microRNA-1290, microRNA-520c-5p, microRNA-1207-5p,microRNA-513a-5p, microRNA-302a, microRNA-675, microRNA-886-5p,microRNA-1297, microRNA-1228) and/or one or more of the down-regulatedmicroRNAs of FIG. 9 (microRNA-505, microRNA-155, microRNA-1261,microRNA-93, microRNA-181a-2, microRNA-181a, microRNA-342-5p,microRNA-21, microRNA-339-3p, microRNA-150, microRNA-17, microRNA-181b,microRNA-501-3p, microRNA-27a, microRNA-23a, microRNA-99b,microRNA-342-3p, microRNA-181a, let-7e, microRNA-769-5p, microRNA-320c,microRNA-1260, microRNA-766, microRNA-339-5c, microRNA-let-7b,microRNA-432, microRNA-125a-5p, microRNA-320a, microRNA 181c,microRNA-140-3p, microRNA-500, microRNA-146a) differs from that of acontrol quantity by a factor of 2 or greater or higher in otherembodiments, for instance by a factor of 3 or greater, a factor of 4 orgreater, or a factor of 5 or greater in some embodiments.

Following determination that the concentration of one or more leukocytemiRNAs in a test sample differs from that of a control concentration bya factor of about 2 or more, a method can also include treating thesubject from whom the sample was derived for endometriosis.

The specific treatment utilized for endometriosis can vary dependingupon the medical history of the patient, the presence of symptoms and,if so, the nature of the symptoms, the extent of the disease state, andso forth.

Analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs) can beeffective treatment in some cases to relieve pain resulting fromendometriosis. Examples of analgesics and/or NSAIDs as may beincorporated in a treatment protocol can include analgesics availableover the counter (OTC) at relatively low dosages including acetamide(acetaminophen or paracetamol), acetylsalicylic acid (aspirin),ibuprofen, ketoprofen, naproxen and naproxen sodium, and the like.Prescription analgesics and/or anti-inflammatories as may beincorporated in a treatment protocol may include, without limitation,OTC analgesics at concentrations requiring a prescription, celecoxib,sulindac, oxaprozin, salsalate, piroxicam, indomethacin, etodolac,meloxicam, nabumetone, keteroloc and ketorolac tromethamine, tolmetin,diclofenac, diproqualone, and diflunisal. Narcotic analgesics mayinclude codeine, hydrocodone, oxycodone, fentanyl, and propoxyphene.

A treatment protocol may include one or more steroidal anti-inflammatorycompounds, primarily glucocorticoids, including, without limitation,cortisone, dexamethasone, prednisolone, prednisone, hydrocortisone,tramcinolone, and methylprednisolone, betamethasone, and aldosterone.

Hormonal therapies can also be utilized in treatment of endometriosis,often in conjunction with one or more analgesics. Endometrial tissuerequires estrogen to grow, and itself produces estrogen. Thus hormonaltherapies that lower estrogen levels and/or increase levels of hormonesthat counteract effects of estrogen can be effective. Oralcontraceptives, for example, can reduce or eliminate pain arising fromendometriosis. Progestins and androgens (natural or, more commonly,synthetic) can interfere with the monthly cycle of endometrialthickening, breakdown and bleeding, The most widely used progestin,medroxyprogesterone, is typically administered as a depot injection.Androgens, including danazol(17α-ethynyl-17β-hydroxy-4-androsteno[2,3-d]-isoxazole) and gestrinone(ethylnorgestrienone), can be administered orally.

Hormonal therapy can include use of drugs that interact withgonadotropin releasing hormone (GnRH), whether as agonists (e.g.,nafarelin or leuprolide) or antagonists, and further can include use ofaromatase inhibitors that block conversion of androgens such asandrostenedione and testosterone to estrogen.

Hormonal therapies can be administered systemically, for example orally,intranasally or by injection in dosages as known in the art, Danazol,for example, can be administered orally at a daily dose of about 200 mgto about 800 mg for a treatment period of 3-6 months, occasionallyextended to 9 months.

In some embodiments, pharmaceutical compositions and delivery systemscan include intravaginal administration of androgens such as gestrinoneand danazol. For example, gestrinone can be administered by a vaginalroute to women with endometriosis so as to reduce dyspareunia anddysmenorrhea with lower side effects such as seborrhea, acne and weightgain as compared to oral administration. Intravaginal administration caninclude utilization of a vaginal ring or vaginal suppositories as knownin the art.

Hormonal therapies can be formulated for administration as a gel, cream,powder, tablet, suppository, capsule, liquid, suspension, dispersion,ointment, foam, etc. formulation and can include additives as aregenerally known in the art in association with the active agents for theadministration route of choice. Additives can include, by way of exampleand without limitation, emulsifiers, wetness agents, polymeric carriers(e.g., biodegradable polymeric carriers), sustained release carriers(hyaluronic acid, alginates, carboxymethyl cellulose, hydroxyethylcellulose, etc.), salts, excipients, dispersions, buffers, and so forth.Preparations for hormonal therapy as may be utilized in treatment ofendometriosis have been described, for example, in U.S. Pat. No.4,997,653 to Igarashi, U.S. Pat. No. 5,993,856 to Ragavan & DiPiano, andU.S. Patent Application Publication No. 2003/0180356 of Kirschner etal., all of which are incorporated herein by reference.

Treatment can also include surgery in those cases in which furtherexamination or development following determination of dysregulation ofleukocyte microRNAs includes the discovery of lesions, cysts, adhesions,and so forth that may be present in endometriosis. The goal of surgicaltreatment is to excise or coagulate all visible endometriotic peritoneallesions, endometriotic ovarian cysts, deep rectovaginal endometriosisand associated adhesions, and to restore normal anatomy. Laparoscopy canbe used in some cases, which can decrease cost, morbidity, and the riskof adhesions postoperatively.

Endometriosis lesions can be removed during laparoscopy by excision,coagulation or vaporization by laser (carbon dioxide laser,potassium-titanyl-phosphate laser or argon laser), in one embodiment,laparoscopic uterine nerve ablation (LUNA) can be utilized. Superficialovarian lesions can be coagulated or vaporized in one embodiment, Smallovarian endometriomata (e.g., less than about 3 cm diameter) can beaspirated, irrigated, and inspected for intracystic lesions. Theinterior wall can be coagulated or vaporized to destroy the mucosallining. Larger ovarian endometriomata (e.g., greater than about >3 cm)can be removed completely). Ovarian cystectomy can also be a treatmentoption in some embodiments.

Radical procedures such as oophorectomy or total hysterectomy can alsobe considered, but generally only in severe cases.

Through incorporation of the disclosed diagnostic approaches intreatment of endometriosis, earlier detection and diagnosis of thedisease can be possible, which can provide a route to more successfuluse of less invasive treatment options, such as treatment by use ofanalgesics and/or hormone therapy, and avoidance of more invasivetreatments, such as surgical approaches.

While the subject matter has been described in detail with respect tothe specific embodiments thereof, it will be appreciated that thoseskilled in the art, upon attaining an understanding of the foregoing,may readily conceive of alterations to, variations of, and equivalentsto these embodiments. Accordingly, the scope of the present disclosureshould be assessed as that of the appended claims and any equivalentsthereto.

What is claimed is:
 1. A method for diagnosing and treatingendometriosis in a subject comprising: analyzing a test sample to obtaina quantity of a leukocyte microRNA present in the test sample; comparingthe quantity of the leukocyte microRNA in the test sample to a controlquantity of the leukocyte microRNA, wherein a determination that thequantity of the leukocyte microRNA in the test sample differs from thecontrol quantity of the leukocyte microRNA by a factor of about 2 orgreater is indicative of endometriosis in the subject; and administeringa treatment for the endometriosis to the subject.
 2. The method of claim1, wherein the test sample is analyzed to obtain a quantity of geneticmicroRNA, primary microRNA, precursor microRNA and/or mature microRNA.3. The method of claim 1, wherein the leukocyte microRNA isoverexpressed in the test sample.
 4. The method of claim 3, wherein theleukocyte microRNA comprises microRNA-1246, microRNA-1225-5p,microRNA-223, microRNA-451, microRNA-572, microRNA-143,microRNA-219-1-3p, microRNA-92b, microRNA-148a, microRNA-450a,microRNA-593, microRNA-510, microRNA-1224-5p, microRNA-1290,microRNA-520c-5p, microRNA-1207-5p, microRNA-513a-5p, microRNA-302a,microRNA-675, microRNA-886-5p, microRNA-1297, or microRNA-1228, orcombinations thereof.
 5. The method of claim 4, wherein the leukocytemicroRNA comprises microRNA-1225-5p, microRNA-572, microRNA-219-1-3p,microRNA-675, or microRNA-886-5p or combinations thereof.
 6. The methodof claim 1, wherein the leukocyte microRNA is underexpressed in the testsample.
 7. The method of claim 6, wherein the leukocyte microRNAcomprises microRNA-505, microRNA-155, microRNA-1261, microRNA-93,microRNA-181a-2, microRNA-181a, microRNA-342-5p, microRNA-21,microRNA-339-3p, microRNA-150, microRNA-17, microRNA-181b,microRNA-501-3p, microRNA-27a, microRNA-23a, microRNA-99b,microRNA-342-3p, microRNA-181a, let-7e, microRNA-769-5p, microRNA-320c,microRNA-1260, microRNA-766, microRNA-339-5c, microRNA-let-7b,microRNA-432, microRNA-125a-5p, microRNA-320a, microRNA-181c,microRNA-140-3p, microRNA-500, or microRNA-146a, or combinationsthereof.
 8. The method of claim 7, wherein the leukocyte microRNAcomprises microRNA-150, microRNA-146a, microRNA-320a, microRNA-505, orlet-7e, or combinations thereof.
 9. The method of claim 1, wherein thestep of analyzing the test sample to obtain the quantity of theleukocyte microRNA present in the test sample comprises ahybridization-based method.
 10. The method of claim 1, wherein the stepof analyzing the test sample to obtain the quantity of the leukocytemicroRNA present in the test sample comprises a quantitative real-timePCR method.
 11. The method of claim 1, wherein the step of analyzing thetest sample to obtain the quantity of the leukocyte microRNA present inthe test sample comprises concentrating or isolating the microRNA fromthe test sample.
 12. The method of claim 1, wherein the treatmentcomprises administration of an analgesic.
 13. The method of claim 1,wherein the treatment comprises administration of one or more hormones.14. The method of claim 1, wherein the treatment comprises a surgicaltreatment.
 15. A method for diagnosing and treating endometriosis in asubject comprising: analyzing a test sample to determine theoverexpression in the subject of one or more cytokines selected fromIL-17, IFN-α, IL-4, and 1L-10 or combinations thereof, the analysiscomprising determining the quantity of a leukocyte microRNA present inthe test sample, the leukocyte microRNA being microRNA-150a,microRNA-146a, microRNA-505, microRNA-320a, or let-7a microRNA precursoror combinations thereof; comparing the quantity of the leukocytemicroRNA in the test sample to a control quantity of the leukocytemicroRNA, wherein a determination that the leukocyte microRNA in thetest sample is underexpressed by a factor of about 2 or greater ascompared to the control quantity of the leukocyte microRNA is indicativeof endometriosis in the subject; and administering a treatment for theendometriosis to the subject.
 16. The method of claim 15, wherein thetest sample is analyzed to determine a quantity of genetic microRNA,primary microRNA, precursor microRNA and/or mature microRNA.
 17. Themethod of claim 15, wherein the step of analyzing the test sample toobtain the quantity of the leukocyte microRNA present In the test samplecomprises a hybridization-based method.
 18. The method of claim 15,wherein the step of analyzing the test sample to obtain the quantity ofthe leukocyte microRNA present in the test sample comprises aquantitative real-time PCR method.
 19. The method of claim 15, whereinthe step of analyzing the test sample to obtain the quantity of theleukocyte microRNA present in the test sample comprises concentrating orisolating the microRNA from the test sample,
 20. The method of claim 15,wherein the treatment comprises administration of an analgesic.
 21. Themethod of claim 15, wherein the treatment comprises administration ofone or more hormones.
 22. The method of claim 15, wherein the treatmentcomprises surgical treatment.